Figure 7

Hsc70 inhibits Apoptin-mediated NF-κB p65 gene down-regulation. The mRNA level of chicken NF-κB p65 in DF-1 cells was determined by semi-quantitative RT-PCR. Chicken β-actin mRNA was normalized to the RT-PCR. (A) Apoptin inhibits NF-κB p65 gene expression. DF-1 cells were transfected with pCMV-Myc-Apoptin plasmids or pCMV-Myc controls. Twenty-four hours after transfection, RNA were prepared and subjected to Semi-quantitative RT-PCR analysis. (B) The relative levels of NF-κB p65 mRNA. The density of bands in (A) was quantitated by densitometry. The relative level of NF-κB p65 mRNA was calculated as follows: the band density of NF-κB p65 mRNA/the band density of β-actin mRNA. (C) Effects of Hsc70 RNAi on the expression of endogenous Hsc70. DF-1 cells were transfected with Hsc70 RNAi (#1 and #2) or controls. Seventy-two hours after transfection, cell lysates were analyzed by Western blot with anti-Hsc70 monoclonal antibody. β-actin was used as an internal control. (D) Knockdown of Hsc70 inhibited the Apoptin caused down-regulation of NF-κB p65. DF-1 cells were transfected with the #2 Hsc70 RNAi or controls. Hsc70 RNAi treated cells were transfected with pCMV-Myc-Apoptin plasmids. Twenty-four hours after transfection, cells were lysed and subjected to semi-quantitative RT-PCR analysis. (E) The relative levels of Apoptin-mediated gene expression of NF-κB p65 in Hsc70 RNAi treated cells vs RNAi controls. The relative level of NF-κB p65 mRNA was determined as in (B).